Sequences from an endosymbiont and their uses

ABSTRACT

rDNA corresponding to an endosymbiotic bacteria associated with  Ecteinascidia turbinata  has been identified. The bacterium appears to be responsible for the biosynthesis of ecteinascidin compounds. The 16S rDNA sequence corresponding to  Candidatus  Endoecteinascidia frumentensis SEQ ID NO: 1 has been deposited in GeneBank with the accession number AY054370.

FIELD OF THE INVENTION

The present invention relates to polynucleotide sequences isolated froma bacterial endosymbiont of the ascidian Ecteinascidia turbinata,microorganisms containing them and their use in the production ofecteinascidin compounds. The present invention further relates topolypeptides or proteins encoded by said polynucleotide sequences andtheir use in the production of ecteinascidin compounds.

BACKGROUND TO THE INVENTION

The ascidian, Ecteinascidia turbinata, is a colonial tunicate from thefamily Perophoridae, found in the Caribbean and Mediterranean Seas. Asan important component of the benthic ecology of the Caribbeanmangroves, it has been the subject of various studies examiningsettlement, species succession and larval behaviour.

In the 1960's interest in this species was heightened when an extract ofthe animal was found to have cytotoxic properties. It was not until the1980's that the compounds conferring these properties, theecteinascidins, were identified and characterised. One of thesecompounds, ET-743, with remarkable cytotoxic acitvity, is presently inclinical trials for the treament of cancer.

Nearly 20 years on, little is known about the production of theseimportant secondary metabolites or what function, if any, they play inthe animal.

Bioactive secondary metabolites are found in many marine invertebrates;especially sponges, molluscs, bryozoans and ascidians, and new compoundsare constantly being described.

Marine invertebrate secondary metabolites encompass a wide range ofchemical types including macrolides, terpenes, steroids, peptides andalkaloids, frequently with complex structures. As yet, there is littledirect evidence for the function these compounds may have in theirhosts, although relevant laboratory and field-based studies arebeginning to address this area. Some investigations support a role inchemical defense, as anti-fouling, anti-infective or anti-predationagents. Many sponges are known to produce noxious chemicals and feedingstudies have indicated that these compounds appear to confer protectionagainst predators. Marine larvae of chemically defended adults alsopossess anti-predatory compounds.

As soft bodied, sessile marine invertebrates, adult ascidians areespecially susceptible to predation, and to fouling of their externalsurfaces. The production of potentially defensive secondary metabolitesappears widespread among certain groups of ascidians. Feeding studiesusing crab and fish predators have indicated that in some ascidiansthese metabolites confer antipredatory protection. Many ascidians alsoproduce large, conspicuous larvae, which develop in a brood pouch. Theselarvae are released in daylight hours, so they can search for optimalsettlement sites, and have a short swimming phase. This strategy leavesthe larvae exposed to predators and it has been suggested that selectionby these predators may favour the evolution of distasteful larvae.Indeed many such larvae may be chemically defended, being unpalatable topredators when presented in feeding experiments.

There is little known of the mechanisms of production of secondarymetabolites in many species. There is evidence to support the theorythat symbionts (especially bacterial ones) within the invertebrate hostproduce at least some of these secondary metabolites, either on theirown, or in conjunction with their host. This idea is based on the factthat some secondary metabolites show close similarities to compoundsproduced by bacteria. Although the evidence to support this theory isstill limited, and in some cases the picture is likely to be verycomplex with no clear unique source for a metabolite, certain studieshave added experimental support to the argument.

Although there are many secondary metabolite-producing ascidians, thereare very few studies on the presence of microorganisms specificallyassociated with ascidians, as epi- or endobionts. However, there are nowmany descriptions of symbiotic associations between bacteria and othermarine invertebrate hosts, for example in sponges, molluscs, bryozoansand echinoderms.

Despite the increasing number of studies, the association betweenchemical defense, secondary metabolites and bacterial symbiosis, is notunderstood.

Ecteinascidia turbinata is the source of natural product ET-743 which isbeing developed as antitumoral agent. The cytotoxic compound isextracted from the ascidian which is obtained by aquaculture. Due to thelife cycle of the Ecteinascidia turbinata this is a time consuming andlaborious process.

Chemical routes to ET-743 are the total synthesis, and the hemisynthesisfrom cyanosafracin B obtained by fermentation. Although these processesprovide alternatives to the natural sources of ecteinascidins, theystill involve numerous chemical steps and are expensive.

There is a need to provide new sources of the ecteinascidin compoundswhich are not subject to the difficulties described above.

SUMMARY OF THE INVENTION

The present invention is directed to a DNA sequence as defined by SEQ IDNO: 1 below or fragments (parts) thereof. SEQ ID NO: 1ATGAATTCTGGTGGCACTGCTTAACACATGCAAGTCGAACGGTAACATAATAAATGTTTTTTACATTTATGGATGACGAGTGGCGGACGGGTGAGTAACGCGTAGGAACCTACCTTTTAGTGGGGGATAGCAGTGGGAAACTACTGGTAATACCGCATGATACTTTAGAGTTAAAACTAGCTGAATTTTATAGCTTGTGCTAAAAGACGGGCCTGCGTTAGATTAGCTTGTTGGTAAGGTAACGGCTTACCAAGGCAACGATCTATAGCTGTTCTGAGAGGAAGATCAGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGGAAGCCTGATCCAGCAATGCCACGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTATTAGCGAAGAAGATATAATGGTTAAGAGCTTAATATATTTGACGTTAGCTAAAGAAAAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTATTGGGCGTAAAGAGCCTGTAGGTGGATAATTAAGTCAGATGTGAAATCCCAAAGCTTAACTTTGGAACTGCATTTGAAACTAATTATCTAGAGTATAGTAGAGGGTAGAGGAATTTCCGGTGTAGCGGTGAAATGCGTAGAGATCGGAAGGAACATCAGTGGCGAAGGCGTCTACCTGGGACTAAAACTGACACTGAGAGGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACTATGAGTACTAACTGTTGGAATTTTTAAATTTTAGTAGTGGAGCTAACGCAATAAGTACTCCGCCTGGGGATTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCTACTCTTGAAATCCTTCGTACTTTATAGAGATATAAAGGTGCCTTTGGAACGAAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTTCCCTTAGTTGCCAGCGTGTAAAGACGGGGACTCTGAGGGGACTGCCGGTGATAAACCGGAGGAAGGCGAGGACGACGTCAAGTCATCATGGTCCTTACGAGTAGGGCTACACACGTGCTACAATGGTATGTACAAAGGGAGGCAAAATTGTAAAATCTAGCAAATCCCCAAAAGCATATCTTAGTCCGGATTGAAGTCTGCAACTCGACTTCATGAAGTTGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGAAGTGGAATGCACCAGAAGTGGCTAGGATAACCGAAAGGAGTCCGGTCCCTACGGTGTGTTTCGTAACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGAACT GCFragments (parts) of the sequence suitably have a length of at least 5,10, 15, 20, 25, 30 or more nucleotide residues. The invention alsoembraces modifications or variations in the sequence SEQ ID 1, includingdeletions, insertions, replacements and other changes. Such modified orvaried sequences typically have at least 50%, 70%, 75%, 85%, 90%, 95% or97% homology with SEQ ID NO:1. References in the following descriptionto the SEQ ID NO: 1 or fragments (parts) thereof include suchmodifications or variations.

In another aspect, the present invention relates to the use of the DNAsequence SEQ ID 1, or parts thereof, in an assay to identify nucleicacid molecules involved in the biosynthesis of ecteinascidin compounds,particularly by walking chromosome techniques.

The present invention is also directed to (a) polynucleotide sequencesisolated from Ecteinascidia turbinata in any of its development phases,its dissociated cells or cultures thereof, and/or a microorganismassociated therewith, involved in the biosynthesis of ecteinascidincompounds, identified with an assay as defined above using the sequenceSEQ ID NO: 1 or parts thereof.

The invention is also directed to (b) a sequence which is able tohybridize, under stringent conditions, with a molecule according to (a),or fragments (parts) thereof. Suitable conditions for such hybridisationare given in the examples, at 45° C. for 3 hours in buffer (0.9M NaCl,20 mM Tris-HCl pH 7.2, 1× Denhardts, 0.1% SDS, 5 mM EDTA, 0.1 mg/mlPoly(A)) with 2.5 ng/μl of probe which had been reconstituted in TE.

The invention is also directed to sequences which, because of thedegeneracy of the genetic code, differ from the molecules according to(a) and (b), or parts thereof.

A polynucleotide, modification, variant or fragment of the invention maybe single stranded or double stranded and may be DNA, RNA or a DNA/RNAhybrid.

In another aspect the present invention relates to polypeptides orproteins encoded by the DNA or other polynucleotide molecules as definedabove.

A modified or variant polypeptide suitably has at least one biologicalfunction of the non-modified or non-variant polypeptide or protein. Itis preferred that modification or variation of a polypeptide or proteinof the invention is such that a biological function of the polypeptideor protein, in particular a function relating to biosynthesis of anecteinascidin compound, precursor, intermediate, or a compound involvedin biosynthesis thereof, is modulated, maintained or improved.

The term polypeptide is interchangeable with protein. A polypeptide orprotein and a modified or variant polypeptide or protein arestructurally related in terms of amino acid composition and sequence.Structurally related polypeptides have at least 60%, preferably at least70%, more preferably at least 80%, yet more preferably at least 90%,further preferably at least 95% amino acid sequence homology. A modifiedor variant polypeptide or protein may be chemically modified or may haveone or more amino acid as substitution, deletion and/or addition.Preferably 1 to 20, 1 to 16, 1 to 12, or 1 to 10 amino acids aresubstituted, deleted and/or added; most preferably 1, 2, 3, 4, 5, or 6amino acids are substituted, deleted and/or added. Preferably, themodification of the polypeptide is by amino acid substitution, which canbe substitution of one or more amino acids, preferably by substitutionof 1 to 20, 1 to 16, 1 to 12, or 1 to 10 amino acids, more preferablysubstitution of 1, 2, 3, 4, 5, or 6 amino acids. Alternatively,modification of the polypeptide may be by deletion of one or more aminoacids, preferably by deletion of 1 to 20, 1 to 16, 1 to 12, or 1 to 10amino acids; more preferably by deletion of 1′ 2, 3, 4, 5, or 6 aminoacids.

In another aspect the present invention is also directed to amicroorganism comprising a sequence according to SEQ ID NO: 1 or partsthereof. More particularly the microorganism is a bacterium.

In another aspect, the invention is directed to the use of amicroorganism as defined above in the production of an ecteinascidincompound, a precursor or intermediate thereof, or a compound involved inthe biosynthesis of ecteinascidin compounds.

The invention furthermore relates to a (host) cell comprising any one ofthe above-described DNA molecules, in particular a (host) cell which istransformed or transfected with any one of the above-described DNAmolecules.

In another aspect the invention relates to a process for theamplification of a DNA molecule which is as described above, preferablyamplification is by PCR amplification.

The invention furthermore relates to a process for investigating thegene cluster for biosynthesizing ecteinascidin compounds, characterizedin that:

-   -   a) hybridization probes which are derived from the DNA sequence        as defined by SEQ ID NO: 1 are prepared and    -   b) these hybridization probes are used for the genomic screening        of DNA libraries obtained from Ecteinascidia turbinata, and    -   c) the clones which are found are isolated and characterized.

DESCRIPTION OF THE FIGURES

FIG. 1. RFLP analysis of clone inserts. HaeIII digests of 16S rDNAinsert. In both cases Lane 1=DNA marker.

A. RFLP's from larval material showing the common Type 1 (lanes 2-5, 7,9, 12), III (lane 10) and IV (lane 11) RFLP pattern.

B. RFLP's from stolon material showing the Type I (lanes 4, 6-8, 10),III (lane 12), IV (lane 5) and VI (lanes 3, 9, 11) patterns. Otherpatterns observed are indicated by Roman numerals.

FIG. 2. Neighbour-joining analysis of 1288 bp from a total of 1451 bp ofunambiguously aligned 16S rRNA gene sequences of Type-I (CandidatusEndoecteinascidia frumentensis) and IV with representative members ofthe gamma-Proteobacteria and an alpha-Proteobacteria outgroup. Thevalues (>50%) at the nodes show the bootstrap support based onneighbour-joining analysis of 100 re-sampled data sets. The scale barrepresents 0.01 substitutions per nucleotide position. Phylogeneticaffiliations, based on RDP II assignments, are shown to the right of thetree. GenBank accession numbers are shown alongside each representativeorganism.

FIG. 3. Neighbour-joining analysis of 1289 bp from a total of 1417 bp ofunambiguously aligned 16S rRNA gene sequences of Type-III and VI withrepresentative members of the Gram positive low G+C % Mycoplasma andBacilli as an outgroup. The values (>50%) at the nodes show thebootstrap support based on neighbour-joining analysis of 100 re-sampleddata sets. The scale bar represents 0.1 substitutions per nucleotideposition. Phylogenetic affiliations, based on RDP II assignments, areshown to the right of the tree. GenBank accession numbers are shownalongside each representative organism.

FIG. 4. Cytochemical and fluorescent staining of E. turbinata cells.Cells of dissociated buds were stained with several dyes (A: Hemacolor.B and C: Hoechst. D: Sytox) and fixed sections of adult zooids werestained with DAPI. Samples stained with fluorochromes were visualisedunder a fluorescent microscope. Fluorescent inclusions are visible withHoechst, Sytox and DAPI (arrows). In some cases the cell nucleus alsoappears stained (arrowheads). Bars=10 μm.

FIG. 5. Sections of E. turbinata larvae.

-   -   A: A section through a whole larva stained with haematoxylin and        eosin. Major anatomical features are visible: s—siphons, t—tail,        b—developing branchial basket, g—gut. Cells which hybridise with        probe Eub338 are often located within the developing branchial        area. B to E: In situ hybridisation of larval tissue width the        probe EUB338. Fluorescent cells indicate areas of probe binding        (arrows).    -   B: A cluster of cells in the branchial basket with fluorescent        inclusions.    -   C: Three cells from a similar area to (B).    -   D: Individual fluorescent inclusions are visible in this host        cell.    -   E: Hybridised cells from a developing larva. Bars (B−E)=10 μm.        Bar (A)=250 μm

FIG. 6. In situ hybridisation of stolon tissue with the EUB338 probe,NonEUB338 probe and EFRU-F2 specific probe (A to C) and in situhybridisation of dissociated bud cells with the EFRU-R1 probe (D-E),using alkaline phosphatase visualisation. A dark brown/black depositshows area of probe binding.

-   -   A: Several cells show binding of the EUB338 probe (arrows).    -   B: The NonEUB338 probe shows a very low level of background        binding.    -   C: The EFRU-F2 probe appears to be binding to a similar cell        type (arrows).    -   D: Similar cells are positive on bud tissue with EFRU-R1 probe.    -   E: two cells showing the positive content as round-shaped        elements. Bars=10 μm.

FIG. 7. Electron microscopy of larval tissue.

-   -   A: E. turbinata larval cell (‘bacteriocyte’) showing numerous        putative bacterial inclusions (arrows). N=nucleus of host cell.        Bar=2 μm.    -   B: High power image of these inclusions showing membrane detail.        Bar=1 μm

DETAILED DESCRIPTION OF THE INVENTION

The present inventors directed their efforts to analyse the bacterialflora associated with Ecteinascidia turbinata, and identify the mostcommonly occurring types both in larvae and adult tissues. Thesebacteria provide a potentially tractable (easily handled) model organismfor examining this association as its secondary metabolites are wellcharacterised and its larvae are known to be chemically defended. Thepresence of specifically associated bacteria, potentially an importantfactor in the chemical defense, provides an alternative source ofecteinascidin compounds.

Molecular methods were employed to ensure that all associated bacteriawere represented, not just those which could be grown by conventionalbacterial isolation and culture techniques. In situ hybridisationanalysis using probes to 16S rRNA was carried out on larval, stolon,zooid and bud tissues of E. turbinata in an attempt to providelocalisation information, in order to confirm the specificity of theassociated bacteria and to identify potentially symbiotic strains.

Surprisingly we identified rDNA corresponding to an endosymbioticbacteria, this bacteria appears to be responsible for the biosynthesisof ecteinascidin compounds.

The 16S rDNA sequence corresponding to Candidatus Endoecteinascidiafrumentensis SEQ ID NO: 1 has been deposited in GeneBank with theaccession number AY054370. The sequences derived from the other twostrains associated with E. turbinata were deposited in GenBank, with therelease of data withheld until the time of publication. SEQ ID NO: 1ATGAATTCTGGTGGCACTGCTTAACACATGCAAGTCGAACGGTAACATAATAAATGTTTTTTACATTTATGGATGACGAGTGGCGGACGGGTGAGTAACGCGTAGGAACCTACCTTTTAGTGGGGGATAGCAGTGGGAAACTACTGGTAATACCGCATGATACTTTAGAGTTAAAACTAGCTGAATTTTATAGCTTGTGCTAAAAGACGGGCCTGCGTTAGATTAGCTTGTTGGTAAGGTAACGGCTTACCAAGGCAACGATCTATAGCTGTTCTGAGAGGAAGATCAGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGGAAGCCTGATCCAGCAATGCCACGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTATTAGCGAAGAAGATATAATGGTTAAGAGCTTAATATATTTGACGTTAGCTAAAGAAAAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTATTGGGCGTAAAGAGCCTGTAGGTGGATAATTAAGTCAGATGTGAAATCCCAAAGCTTAACTTTGGAACTGCATTTGAAACTAATTATCTAGAGTATAGTAGAGGGTAGAGGAATTTCCGGTGTAGCGGTGAAATGCGTAGAGATCGGAAGGAACATCAGTGGCGAAGGCGTCTACCTGGGACTAAAACTGACACTGAGAGGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACTATGAGTACTAACTGTTGGAATTTTTAAATTTTAGTAGTGGAGCTAACGCAATAAGTACTCCGCCTGGGGATTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCTACTCTTGAAATCCTTCGTACTTTATAGAGATATAAAGGTGCCTTTGGAACGAAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTTCCCTTAGTTGCCAGCGTGTAAAGACGGGGACTCTGAGGGGACTGCCGGTGATAAACCGGAGGAAGGCGAGGACGACGTCAAGTCATCATGGTCCTTACGAGTAGGGCTACACACGTGCTACAATGGTATGTACAAAGGGAGGCAAAATTGTAAAATCTAGCAAATCCCCAAAAGCATATCTTAGTCCGGATTGAAGTCTGCAACTCGACTTCATGAAGTTGGAATCGCTAGTAATCGCGAATCGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGAAGTGGAATGCACCAGAAGTGGCTAGGATAACCGAAAGGAGTCCGGTCCCTACGGTGTGTTTCGTAACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGAACTG C

The present invention provides one of the first reports of endosymbioticbacteria in an ascidian. The combination of 16S rRNA gene analysis, insitu hybridisation and electron microscopy, provides evidence for aspecific association between these bacteria and the ascidian host cells.The observation of identical bacteria strains, with similar levels ofdominance, and of similar cell types, in both adult (zooid and stolon)and larval tissues provide strong indications that these cells are aconsistent and important feature of Ecteinascidia turbinata. In situhybridisation based on the obtained sequence data suggest that theCadidatus Endoecteinascidia frumentensis (Type I strain) is the observedsymbiont. Besides that, we found the presence of this bacterial strainin all the development stages and in samples collected all over theworld, and the absence of it in a related tunicate from the samegeographic area.

16S rDNA sequence analysis of dominant bacterial strains in E. turbinatahas identified three novel strains. Candidatus Endoecteinascidiafrumentensis falls in the gamma-proteobacteria subdivsion. Although itis only distantly affiliated to any other known strains within thisgroup, it bears similarities to species in the Legionella andOceanospirillum subgroups. Many previously described marineendosymbionts belong to the gamma-proteobacteria subdivision, withrepresentatives from sponges, oligochaetes and bryozoans covering arange of ecological niches and putative activities.

The spiroplasm-like Type III and VI-symbionts appear to represent a newsub group. Mycoplasms are often found as parasitic organisms invertebrates and invertebrates, although there is increasing evidence tosupport both commensal and saprophytic roles. There are few records ofthem in marine invertebrates, although they have been identified in abryozoan. The significance of finding a Gram negative bacteria (thegamma-proteobacterium), together with two common Gram positive strains(the spiroplasms), in E. turbinata is not known, but it is interestingto note that a similar situation occurs in aphids. Localisation of themycoplasmas in E. turbinata would be a valuable step forward.

The use of molecular techniques to survey bacterial populations hasallowed comprehensive analysis of bacterial complement, overcomingselection for bacteria that are culturable by standard methods. Use of16S rRNA gene sequences to identify bacteria is now the standardprocedure for analysis of bacteria in environmental samples and hasenabled much of the current progress in the area of bacterial symbiosis.Whilst being suitable for surveying of bacterial strains, this techniquecan lead to misinterpretation if attempting to quantify those typespresent and analysis of results must take note of these possiblefailings. For example, it has been found that templates containingGC-rich combinations in the priming sites, might be preferentiallyamplified.

However, the results presented below indicate such a high predominanceof one novel strain, in four different tissues, that it is unlikely thiscould be explained by PCR bias. Similarly, bacterial types possess adifferent number of copies of the 16S rRNA gene, leading to potentialweighting of amplification results. For example, the spiroplasm-likestrains identified are likely to possess only one copy of the gene,whereas the gamma-proteobacteria strain may contain two or more. In thecase of E. turbinata the clear predominance of strains in all tissuesexamined and in all the locations tested, negate the possibilities ofinterpretation errors.

Cytochemical studies and in situ hybridisation using the universalprobes described in the examples suggests that bacteria are locatedwithin host cells in adult and larval tissue. It appears that thesecells are free, possibly in the blood, and present at higher densitiesin the larvae. Here, they are clustered primarily in the developingbranchial basket This high density of bacteria supports the dominanceobserved in the 16S analysis of larval tissue, and together with in situhybridisation using the probe to the Candidatus Endoecteinascidiafrumentensis sequence, adds weight to the evidence that this strain isthe one identified in situ.

The significance of the high numbers of ‘bacteriocytes’ in the branchialbasket is not clear, as the larvae are not feeding, but it suggests thatthe bacteria may be present at sites of optimal nutrition (gaseous andnutrient exchange) as soon as the larvae has settled and the siphonshave opened. However, it is also possible that a high density in thisarea is a side effect of the developing branchial system and that thebacteriocytes are clustered here due to blind ending blood vessels thatare still growing to form the basket. Higher densities of putativebacteriocytes are also found in the stolon, which becomes engorged withcells as colonies regress. Few cell types are present, suggesting thatcells have become undifferentiated, or are primarily for storage, readyto support the next phase of budding. Buds also show high density ofbacteriocytes, which have probably migrated from the stolon directly.However, labeled cells are much harder to detect in zooids, where tissueis diffuse and cell density low. Still several putative bacteriocyteshave been observed by in situ hybridisation, with similarcharacteristics as before. That is, not anchored, but freelycirculating, possibly in the blood.

The forming branchial basket was the target tissue for TEM studies basedon the in situ hybridisation results. The presence of bacteriocytes ofsimilar size and apparent morphology as the cells illuminated byhybridisation in the branchial basket and in dissociated bud cellssuggests that the TEM results are the same cells as those seen byhybridisation. The bacteria appear to be intracellular symbionts ratherthan pathogens or ingested bacteria. This is suggested from a number ofobservations: i) there is no obvious endocytotic behaviour by the cells;ii) the bacteria appear healthy; iii) there is no obvious pathology ofthe host cell in the smaller bacteriocytes; iv) the presence of so manypolyribosomes suggests a large amount of synthetic activity by the cellthat is intended for internal consumption rather than export; v) thebacteria appear to be of a uniform type and the same type has beenobserved in different individual larvae. A double membrane appears tosurround the bacteria, suggesting that they are gram negative. However,the method of fixation was not optimal and further investigations of thefine structure of the bacteriocytes may help to confirm thisobservation.

The presence of the bacteria in non-feeding larvae strongly suggeststhat they are vertically transmitted through the eggs and larvae at eggformation or during brooding. This transmission mechanism is alsoobserved in other marine species, for example in the ascidian Diplosomasimiles, and in some bivalves. Intraovarial transmission of symbionts isalso common in insects. That the bacteria are endocytotic and likely tobe vertically transmitted also suggests that they are unlikely to beculturable in simple media. Culturing experiments followed by 16S RFLPanalysis of 22 bacterial strains derived from larvae, and 9 from zooids,indicate that bacteria similar to the Type I, III and VI strain did notgrow under standard conditions. The difficulty in culturing symbioticbacteria is a barrier to the further elucidation of their roles andmethods to overcome this problem are needed. Some researchers havemanaged to grow a symbiont of Theonella using specific cultureconditions. Efforts to culture intracellular symbionts have been limitedand attempts to culture host cells have generally failed. Ways ofre-addressing this problem would greatly improve the understanding ofbacterial symbioses in general.

The symbionts of Ecteinascidia turbinata appear to be involved in thebiosynthesis of ecteinascidins. Many marine symbionts are thioautotrophsfrom anoxic environments, oxidising sulphur and fixing CO₂. Indeed acomplete sulphur cycle involving two symbionts has recently beenelegantly demonstrated in a marine oligochaete. In the case of Eturbinata it is possible that utilisation of certain nutrients in apotentially anoxic mangrove environment may be advantageous, although E.turbinata is known to grow and thrive in a range of habitats.

E turbinata larvae and adults are vulnerable to predation and/orfouling, thus production of noxious compounds may act as an effectivedefensive strategy. That the larvae are chemically defended, known tocontain secondary metabolites and also house a large population ofapparently endosymbiotic bacteria may be circumstantial. However, it ispossible that a symbiosis between the ascidian and a secondarymetabolite-producing bacterium may confer this chemical protection.

Therefore, the rDNA sequence of the present invention can be used in anassay to indentify the symbiont and to isolate its DNA, for example withthe widely used walking chromosome techniques. This DNA can beresponsible for the biosynthesis of the ecteinascidin compounds of atleast of bioprecursors or intermediates thereof. Once isolated it can beintroduced in a host cell to provide a microorganism producingecteinascidin compounds, providing an alternative source of thesevaluable compounds.

Accordingly, the invention provides a process for producing anecteinascidin compound, precursor or intermediate thereof, or a compoundinvolved in biosynthesis of an ecteinascidin compound, precursor orintermediate, comprising culturing such a host cell under conditionssufficient for biosynthesis of said ecteinascidin compound, precursor orintermediate thereof, or said compound involved in biosynthesis of anecteinascidin compound, precursor or intermediate.

The process can further comprise recovering the ecteinascidin compound,precursor or intermediate thereof, or compound involved in biosynthesisof an ecteinascidin compound, precursor or intermediate thereof from theculture.

EXAMPLES

Tissue Preparation and Dissection:

Ecteinascidia turbinata colonies were obtained from the MediterraneanSea (Formentera, Spain) and Atlantic Ocean (Cádiz, Spain) andtransferred to a marine aquarium at a water temperature of 24° C. Larvaewere obtained from ripe colonies which were undergoing larval release,or by dissection from these colonies. They were rinsed several times insterile filtered seawater before DNA extraction. Immature zooids(without gonads or larvae) were pinched off the colony at the stolon andincubated in sterile seawater at 24° C. for 24 hours, with one change ofwater. They were then given a final rinse in sterile seawater andprepared for DNA extraction. Stolons from regressing colonies wereremoved and rinsed in sterile seawater. They were gently homogenised toliberate cells and this supernatant was removed to another tube,discarding the larger debris. The cells were spun down and pelleted,ready for DNA extraction. All material was snap frozen in liquidnitrogen for storage at −80° C. Scallop muscle (Chlamys opercuiIars) wasremoved from an animal held in the aquarium, rinsed in sterile seawaterand used as a control.

Example 1 16S rDNA Analysis and RFLPs

DNA Isolation, 16S rDNA PCR Amplification and Cloning:

All DNA manipulations were performed under recognised standardconditions. To ensure all cells, both eukaryotic and prokaryotic, werebroken open, the tissue was homogenised in liquid nitrogen in a sterilemortar and pestle (except the stolon cells which were used directly).DNA was isolated either using a GNOME DNA Isolation kit (BIO101, Vista,Calif.), or with the following protocol. Lysis buffer (100 mM Tris-HCI,10 mM EDTA, 150 mM NaCI, 100 μg/ml RNase A) was added to the frozenpowder and allowed to thaw. The homogenised tissue was transferred to asterile 1.5 ml centrifuge tube, lysozyme was added (200 pg/ml finalconcentration) and the sample incubated for 10 minutes at 37° C.Proteinase K and SDS were added (400 pg/ml and 1% final concentrations,respectively) and the suspension incubated for a further 30 minutes at37° C. DNA was purified by two sequential extractions with Tris-HCI (pH8.0) equilibrated Phenol:Chloroform:Isoamyl alcohol (25:24:1), twicewith Chloroform:Isoamyl alcohol (24:1), ethanol precipitated with sodiumacetate (pH 5.2) and washed with 70% ethanol. The DNA pellet wasair-dried and stored in TE buffer (10 mM Tris-HCI, pH 8.0; 1 mM EDTA) at−20° C.

Two different universal 16S rDNA bacterial PCR set of primers and oneset of specific oligonucleotides for DNA sequences of the CandidatusEndoecteinascidia frumentensis 16S rDNA were used for the amplificationexperiments. The universal primers were: forward, 8-AG(AG) GTT TGATC(AC) TGG CTC AG-27; reverse 1509-G(GT)T ACC TTG TTA CGA CTT-1494(primer position is according to E. coli Weisburg, W. G., Barns, S. M.,Pelletier, D. A. & Lane, D. J. 1999. 16S ribosomal DNA amplification forphylogenetic study. J. Bac. 173, 607-703.) and 16SF1 5′-GAG A(G/C)T TTGATC (A/C/T)TG GCT CAG-3′; 1600R 5′-AAG GAG GTG ATC CAG CC-3 (modifiedfrom Dorsch, M. & Stackebrandt, E. 1992. Some modifications in theprocedure of direct sequencing of PCR amplified 16S rDNA. J. Microbiol.Methods, 16, 271-279), and the specific ones were EFRU-F1, 5′-CGG TAACAT AAT AAA TGT TTT TTA CAT TTA TG-3 and EFRU-R1, 5′-TAT GCT TTT GGG GATTTG CTA GAT T-3′ (this study). A DNA Engine (MJ Instruments, USA)thermocycler, or a Mastercycler personal (Eppendorf, Germany) was used,cycled as follows: 94° C. for 2 min., followed by 30 cycles of 55° C.(30 secs), 72° C. (1 min 15 s) and 94° C. (15 s), and a final elongationstep of 72° C. for 10 minutes. Total bacterial 16S rDNA from theextracted DNA was amplified from several dilutions to obtain optimalresults and a “no-DNA” control was run for each PCR mix. The PCR productwas confirmed by 1% agarose gel electrophoresis and ethidium bromidestaining.

When needed, the PCR products were purified (Millipore Ultrafree-DAcolumns) and ligated into pGEM-T (Promega, USA) overnight at 4° C. to16° C. and then transformed into competent E. coli DH5α (LifeTechnologies, UK). Transformants were spread on LB agar containing 50pg/ml ampicillin, 0.2 mM X-gal and 0.16 mM IPTG for blue-whitescreening, and incubated overnight at 37° C.

RFLP Analysis of Selected Clones and Sequencing:

Putative insert-containing clones (100 clones from each of the larval,zooid and stolon, and 40 from the scallop-control material) wereselected from the E. coli plates by picking with a sterile toothpick andemulsifying into a 50 pl PCR reaction containing M13/pUC universalprimers (forward 5′-GTT TTC CCA GTC ACG AC-3′; reverse 5′-CAG GAA ACAGCT ATG AC-3′) and patched onto LB agar containing ampicillin, X-gal andIPTG (as above) for subsequent plasmid isolation. PCR reactions werecycled as follows: 94° C. for 5 minutes, followed by 30 cycles of 50° C.(30 secs), 72° C. (1 min 30 s), 94° C. (15 secs), with a finalelongation step of 72° C. for 10 minutes. Inserts of the appropriatesize (approximately 1700 base pairs) were confirmed by 1% agarose gelelectrophoresis. Positive PCR products were ethanol precipitated andresuspended in sterile distilled H₂O. Restriction fragment lengthpolymorphism (RFLP) analysis was carried out using the restrictionenzymes HaeIII and HhaI (Promega, USA). The restricted PCR products wereelectrophoresed through a 1× Tris-acetate (40 mM), EDTA (1 mM), 3%wide-range agarose gel (Sigma, USA) and stained with ethidium bromide.Kodak 1-D system and software (Kodak, USA) was used to capture theresultant RFLP patterns. Clones representative of each different patternobserved were isolated and grown up in LB broth with ampicillin (75pg/ml) overnight and stored frozen with 10% glycerol at −80° C. PlasmidDNA was isolated from representative clones for DNA sequence analysisaccording to the manufacturers instructions. (Qiagen Spin Mini-Preps,UK). DNA sequencing was performed using the M13 universal primers andABI BigDye chemistry (PE Applied Biosystems, USA) and analysed on an ABI377 DNA sequencer (PE Applied Biosystems, USA).

Results:

The first approach to the analysis of the microorganisms associated toEcteinascidia turbinata was done using total DNA isolated from adultzooids. Direct sequencing of the PCR amplification fragment obtainedwith this DNA and “universal” eubacterial primers 16SF1(5-GAGA(G/C)TTTGATC(A/C/T)TGGCTCAG-3′) and 1600R(5′-AAGGAGGTGATCCAGCC-3′) (modified from Dorsch & Stackebrandt, 1992)resulted in a clean sequence, indicating the prevalence of thismicroorganism in the tunicate. It is defined above as SEQ ID 1.

With the aim to confirm this result, specific oligonucleotides weredesigned for the Candidatus Endoecteinascidia frumentensis 16S rDNA.These oligonucleotides (EFRU-F1, 5′-CGG TAA CAT AAT A AA TGT TTT TTA CATTTA TG-3′ and EFRU-R1, 5′-TAT GCT TTT GGG GAT TTG CTA GAT T-3′) wereused as primers for the PCR amplification experiments performed withtotal DNA isolated from adult zooids from different locations around theworld (Formentera, Menorca, Túnez, Cádiz, Cuba, Florida, Puerto Rico)and with DNA obtained at different phases of development (stolon,embryos, larvae, buds and adult zooids). An amplification band with theexpected size was obtained in all the cases, and RFLP and sequenceanalysis showed that Candidatus Endoecteinascidia frumentensis waspresent in all those samples. As a control, total DNA from a closelyrelated organism, Ecteinascidia conklini was isolated, and PCRexperiments were performed. No amplification was obtained with theCandidatus Endoecteinascidia frumentensis specific primers, althoughbands of the right size could be seen when universal eubacterial primerswere used (data not shown). Positive results were also obtained when E.turbinata DNA was used (both with a sample collected at the same area asE. conklini was and with a sample from a different location), withuniversal and specific primers.

The major presence of Candidatus Endoecteinascidia frumentensis wasconfirmed by the analysis of the bacterial flora associated with thetunicate. To minimise the contribution of non-specifically associatedbacteria contaminating the total DNA pool, tissue was cleaned as far aspossible before DNA extraction. The incubation of live animals insterile seawater helped to depurate (cleanse) the pharynx and gut of E.turbinata, which would have contained large numbers of non-specificallyassociated bacteria. Near full-length 16S rRNA gene fragments wereamplified from the total DNA extracted from zooid, larval, stolon tissueand the control, scallop adductor muscle. After cloning and RFLPanalysis, percentage dominance was assigned to the patterns observed inthe three tissue types in order to determine any commonly occurringtypes. Four types were identified as occurring in all three tissue typesand which also represented the most abundant RFLP types (see Table 2 andFIG. 1). The Type I pattern was the most common, representing between42-67% of RFLP patterns observed in the three tissue types and thesequence analysis of this Type showed that it actually was CandidatusEndoecteinascidia frumentensis. Types VI, III and IV were the remainingthree most abundant patterns (Table 2, FIG. 1). The scallop control datashowed no Type I (Candidatus Endoecteinascidia frumentensis), III or VIpatterns, but a Type IV pattern was observed. This, together with thesequence analysis, identified Type IV as a commonly occurringmarine-associated bacteria, and unlikely to have a specific relationshipwith E turbinata. TABLE 2 Dominance of RFLP patterns from zooid, larvaland stolon material, and the total number of patterns observed. %dominance of RFLP types RFLP Type in E. turbinata tissue (Pattern) ZOOIDLARVAE STOLON Type I (Candidatus 42.6 55.9 67.0 Endoecteinascidiafrumentensis) Type III 5.6 9.6 5.3 Type IV 7.8 1.0 4.2 Type VI 14.6 4.310.0 Total number of 89 93 94 clones digested Total number of 21 11 ispatterns observed

Example 2 Phylogenetic Analysis

Compiled DNA sequences from two or more independent clones of each RFLPtype sequenced were aligned with the RDP II database (Maidak B L, Cole JR, Lilburn T G, Parker C T Jr, Saxman P R, Farris R J, Garrity G M,Ohsen G J, Schmidt T M, Tiedje, J M. 2001. The RDP-II (RibosomalDatabase Project). Nucleic Acids Res. 29, 173-174) using SEQUENCE MATCHand checked with CHIMERA CHECK to ensure that no sequence was chimeric.Phylogenetic inference of the novel 16S rDNA sequences was achieved byaligning the compiled 16S rDNA sequences with representative sequencesobtained from the RDP II (Madiak et al., 2001) and GenBank usingCLUSTALX (Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &Higgins, D. G. (1997). The ClustalX windows interface: flexiblestrategies for multiple sequence alignment aided by quality analysistools. Nucleic Acids Research 24, 4876-4882.). Alignments were manuallycorrected and ambiguous positions removed. PAUP* 4.0b8 (Swofford, D. L.(2001). PAUP*. Phylogenetic Analysis Using Parsimony (*and OtherMethods). Version 4. Sinauer Associates, Sunderland, Mass.) was used toinfer phylogenetic trees using parsimony and neighbour-joining analysisaccording to the Kimura 2-parameter model of nucleotide substitution(Kimura, M. (1980). A simple method for estimating evolutionary rate ofbase subsitutions through comparative studies of nucleotide sequences.J. Mol. Evol. 16, 111-120) and assuming the among-site rate variationhaving a gamma distribution shape of 0.5. Bootstrap support for theinferred tree was established following re-sampling of 100 data setsbased on neighbour-joining analysis.

Results:

Four near complete 16S rRNA genes of the most commonly occurringbacterial strains were sequenced. Alignment of these sequences with theRDP II database indicated that the 16S rRNA gene sequences of Types I(Candidatus Endoecteinascidia frumentensis), III and VI were unrelatedto any particular bacterial species in the database. The Type IV patternwas shown to have 98% sequence similarity with Pseudomonas fluorescens(D86001). Type I (Candidatus Endoecteinascidia frumentensis) could bereliably assigned as a member of the Gram negative, gamma-Proteobacteria(FIG. 2). Within this group, it showed a closer relationship to theLegionella group. However, the deep divergence and long branch-length ofthis sequence makes this a tentative assignment. This was reinforced byparsimony analysis, which was not able to conclusively support thebranching order as depicted in FIG. 2. The closest sequence affiliationto Candidatus Endoecteinascidia frumentensis was an unidentifiedgamma-Proteobacteria (AB015255) isolated from the sediment of a deep-seatrench. Types III and VI shared 97.3% sequence similarity to each other.Phylogenetically, they were most similar to Spiroplasma group, which aremembers of the low G+C % Gram positive Mycoplasma and relatives (FIG.3). Both Type III and VI, although deeply divergent from within theSpiroplasma group, could be reliably assigned to this group of theMycoplasmas.

Example 3 Cytochemical and Fluorescent Staining

Dissociation of Buds:

Esteinascidia turbinata stolon was obtained from Cádiz (Spain) andtransferred to a marine aquarium at a water temperature of 24° C. Newgenerations of growing buds were collected from the stolon and washedseveral times in sterile filtered seawater. Bud tunic was removed usingforceps under sterile conditions and nude buds (20-30) were washed fourtimes in sterile seawater. Buds were placed in dissociation medium(sterile seawater plus 0.1% collagenase), and incubated for 3 hours at27° C. Bud tissue was dissociated by pipetting. To obtain a suspensionof single cells, sample was left to decant on the bench for 3 minutes.The upper part of the sample was saved to other tube and cellsconcentrated by centrifugation at 1200 rpm/RT/4 minutes. Cells werewashed with 500 μl of sterile seawater, centrifuged again andresuspended in 100 μl of fixation media.

Cell Fixation and Slide Preparation for In Situ Hybridisation of SingleCells:

E. turbinata single cells were fixed at RT for 30 minutes in 100 μl offixation media (4% formaldehyde, 5% acetic acid in sterile seawater).Fixed cells were washed with 500 μl of sterile seawater, resuspended in20 μl of sterile seawater and spread on a glass slide. Slides werepreviously treated with TESPA in order to improve cell attachment.Slides were air dried, washed with sterile seawater and stored in 70%ethanol (5° C.) until performing in situ hybridisation.

Cell Staining

Samples of dissociated and fixated bud cells were stained withHoechst-33342, Dapi, Sytox or Hemacolor, for 15 minutes. Cells werecentrifuged (1200 rpm/RT/4 minutes), resuspended in 250 μl of sterileseawater and observed under UV light in a inverted fluorescentmicroscope (Leica DM IRB).

Results:

Dissociated bud cells or fixed sections of adults were used for severalstaining experiments in order to identify intracellular microorganismsin E. turbinata cells. By using Hemacolor, a general staining method,intracellular positive granules could be observed in some cells ofdissociated buds (FIG. 4A). Several fluorescent dyes specific fornucleic acids were also employed. These dyes are sensitive enough toallow detection of DNA/RNA containing particles as bacteria andmycoplasms, but not subcellular organelles as mitochondria orchloroplasts. Hoechst and Sytox showed intracellular positive elementsin cells of dissociated buds. In some cases, just a few fluorescentround or rod-shaped elements were observed inside large vacuoles (FIG.4B) but also dense stained cells showing a granular appearance could bedetected (FIGS. 4C and D). In fixed sections of E. turbinata zooids,DAPI staining showed the same type of fully stained cells (FIG. 4E). Thecell nucleus is visible in some cases (FIGS. 4A, B and E), when nothidden by fluorescent particles (FIGS. 4C and D).

Example 4 In Situ Hibridisation with a Universal Probe

In situ hybridisation was carried out using standard methods. Tissue wasfixed in 4% Paraformaldehyde (PFA) in TBS (0.1MTris-HCI, 0.9% NaCI) with0.1M MOPS at room temperature for 30 mins to 1 hour. It was thenembedded in paraffin wax, sectioned, and sections were mounted ongelatin-coated slides. Two panels of sections were mounted on eachslide, placing alternate sections on each panel, to provide a test andcontrol panel of the same area of the tissue. The slides were thenprepared for hybridisation. In brief, after rehydration they wererinsed, incubated in 1 μg/ml Proteinase K, post-fixed in 4% PFA in TBSfor 10 minutes, rinsed and treated with 0.25% acetic anhydride in 0.1MTriethanolamine-HCI pH8, dehydrated and air dried, ready for storage at−80° C.

Initial in situ hybridisation studies were carried out using abiotinylated universal bacterial 16S rRNA probe to identify sites ofpotential interest (EUB338 5′-GCT GCC TCC CGT AGG AGT-3′, and a controlprobe NON-EUB338 5′-ACT CCT ACG GGA GGC AGC-3′) (Amann, R. I. Binder, B.J., Olson, R. J., Chisholm, S. W., Devereux, R. & Stahl, D. A. 1990.Combination of 16S rRNA-targeted oligonucleotide probes with flowcytometry for analysing mixed microbial populations. Appl. Environ.Microbiol. 56, 1919-1925.). Other controls consisted of incubations withno probe, and panels of attached bacterial cells (E. coli) which hadbeen fixed and processed as the sections. Hybridisation was carried outat 45° C. for 3 hours in buffer (0.9M NaCI, 20 mM Tris-HCI pH7.2, 1×Denhardts, 0.1% SDS, 5 mM EDTA, 0.1 mg/ml Poly(A)) with 2.5 ng/μl ofprobe which had been reconstituted in TE. Hybridisation chambers withdual ports were used to prevent evaporation (Grace BioLabs: 22 mm×22 mmchamber for each panel of sections on the slide). Followinghybridisation the slides were washed 2×15 minutes in wash buffer (0.9MNaCl, 20 mM Tris-HCI pH 7.2, 0.1% SDS) at 48° C. Binding of probe wasvisualised using Avidin-DN (Vector Labs) as recommended by themanufacturer. Sections were mounted in Vectorshield (Vector Labs) andviewed on a Zeiss Axioskop with fluorescent attachments. Forvisualisation with alkaline phosphatase (AP) the slides were washedafter hybridisation and incubated in AP conjugated anti-biotin antibody(Vector Labs) diluted 1:1000 in buffer (20 mM sodium phosphate, 0.9%NaCI, 0.1% Tween, 0.1% BSA) at 4° C. overnight. They were then washedand visualised using a BCIP/NBT-AP substrate kit (Vector Labs).

Results:

Hybridisation of the universal bacterial EUB 338 16S rRNA probe wasobserved in larval sections (FIG. 5), stolon material and at low levelsin zooid sections. Positive in situ hybridisation was found in cells ofapproximately 10-12 μm diameter which demonstrate a granular appearance.These cells usually had an unstained area at one pole, which was thoughtto be the host cell nucleus. In larvae the cells were predominantlyfound in the developing branchial basket (FIG. 5). In zooid tissue, onlya few positively hybridising cells were observed. These cells werelocated in the region of the pharynx and with cells possibly associatedwith blood vessels. The background fluorescence was relatively high, buthybridisation positive cells were easily distinguishable on the testsections, but were clearly not present in the controls. Dissectedportions of regressing stolon and budding tissue were also examined byfluorescent in situ hybridisation. With these preparations, thebackground fluorescence under the FITC filter was too high to facilitateaccurate interpretation of hybridisation. Consequently, subsequenthybridisation experiments used the histochemical AP visualisationsystem. Hybridising cells were found scattered throughout the stolon andbud tissue. The appearance of the hybridisation-positive cells in thestolon and bud tissue had the same general appearance as observed in thelarvae, with the hybridised part of the cell exhibiting granularstaining and an unstained, approximately polar region, presumablyrepresenting the host nucleus (FIG. 6A). The non-Eub338 probe gave verylow levels of background binding using the AP system, such that it wasalmost indiscernible when compared to the intensity of Eub338 binding(FIG. 6B). In all cases where the control was buffer without probe, thecells hybridised in the test sections could not be observed in thecontrol sections.

Example 5 Specific Probes: Dot Blots and In Situ Hybridisation

Probes were designed to putatively unique regions of the CandidatusEndoecteinascidia frumentensis and Type III 16S rDNA sequences:Oligonucleotide Sequences (5′-3′) Application^(a) References Symbiontspecific EFRU-F1 CGG TAA CAT AAT AAA P, I, S Herein TGT TTT TTA CAT TTATG EFRU-R1 TAT GCT TTT GGG GAT I, D, S Herein TTG CTA GAT T EFRU-R2 CTTTCG GTT ATC CTA I, D Herein GCC AC Domain bacteria Type III-1 GCA ACTATT TCT AGC D Herein TGT TAT TC Type III-4 AGC TTT GCA CTG GAT D HereinGTC AAG EUB338 GCT GCC TCC CGT AGG I, D Amann et al., 1990 AGTNON-EUB338 ACT CCT ACG GGA GGC I Amann et al., 1990 AGC EUB8-f AG(AG)GTT TGA TC(AC) P, S Weiburg et al., TGG CTC AG 1999 EUB1509-r G(GT)T ACCTTG TTA CGA P, S Weiburg et al, CTT 1999 16S-F1 GAG A(G/C)T TTG ATC P, SModified from (A/C/T)TG GCT CAG Dorsch & Stackebrandt, 1992 1600R AAGGAG GTG ATC CAG P, S Modified from CC Dorsch & Stackebrandt, 1992Universal primers M13/pUC-f GTT TTC CCA GTC ACG S AC M13/pUC-r CAG GAAACA GCT ATG S AC^(a)P = PCR primer; I = In situ hybridization probe; D = Dot-blot probe;S = Sequencing primers

Probe specificity was initially examined by submitting the probesequence to the SEQUENCE MATCH and PROBE MATCH on the RDP II database(Maidak et al., 2001) and compared to E. coli 16S secondary structure tocheck for optimal regions of binding (Amann, R. I., Ludwig, W. &Schleifer, K-H. 1995. Phylogenetic identification and in situ detectionof individual microbial cells without cultivation.

Microbiological Reviews. 59, 143-169; Zheng, D., Alm, E. W., Stahl, D.A.& Raskin, L. 1996. Characterisation of universal small-subunit rRNAhybridisation probes for quantitative molecular microbial ecologystudies. Appl. Environ. Microbiol. 62, 4504-4513).

Conditions for probe binding were confirmed by dot blot analysis. 16SrDNA PCR products from the Type I Candidatus Endoecteinascidiafrumentensis, III, IV & VI plasmid clones (50 ng) and E. coli DH5 alphawere spotted onto a positively charged nylon membrane as permanufacturers instructions (Schleicher & Schuell). The membranes wereprehybridised at 46° C. for 1 hour in hybridisation buffer (aspreviously plus 2% Marvel), and then hybridised at 46° C. for 3 hourswith 0.5 ng/μl of probe. Membranes were washed 2×15 minutes at 50° C.(wash buffer as before), blocked in NaPBS (20 mM sodium phosphate, 0.9%NaCl) with 1% BSA for 1 hour and visualised using anti-biotin alkalinephosphatase (1:2000 in NaPBS, 0.1% BSA) and the BCIP/NBT-AP substratekit (Vector Labs).

Following dot blots, hybridisation with EFRU-F2 probe was carried out onsections at 46° C. for 3 hours, in hybridisation buffer, containing 0,5, and 10% formamide, plus a no probe control of each, in an attempt tooptimise the stringency for each probe. 2×15 minute washes were carriedout at 50° C. Slides with control bacteria (E. coli) were also used ateach of the formamide concentrations, with each probe, to further checkfor stringency. Stolon tissue was screened on sections as this provideda more uniform basis for comparisons. Successful hybridisations wererepeated on larval sections.

Results:

The dot blots indicated EFRU-F2 was specific for the Type I (CandidatusEndoecteinascidia frumentensis) sequence, binding only to Type I 16SrDNA and not to that of the other common types isolated from E.turbinata or to E. coli 16S rDNA, under standard hybridisationconditions see Table 3.

Same results were obtained for EFRU-R1 probe. TABLE 3 Dot-blothybridisation of unique 16S rRNA in situ probes Probes 16S rDNA PCRproduct Eub338 EFRU-F2 Type III-1 Type 111-4 Type I X X X — Type III X —X X Type IV X — — — Type VI X — X X E. coli X — — —

Type III-4 was also specific for Type III and VI sequence, but typeIII-I cross-reacted with the Type I DNA under these conditions. TheType. I probe tested on stolon sections bound to similar cells as thosehighlighted by the universal eubacterial probe EUB338, suggesting thatthese bacteria are the Candidatus Endoecteinascidia frumentensissymbiont (FIG. 6C).

In situ hybridisation of EFRU-R1 probe on dissociated bud cells showedthe granular cell type previously described (FIG. 6D) together with someother cells where isolated positive elements could be observed (FIG.6E). These figures could correspond to lightly crushed granular cellsshowing the cellular content. The round shaped elements are suggested tobe putative Type I bacteria (Candidatus Endoecteinascidia frumentensis),which appear tightly packed in certain cell types.

Example 6 Electron Microscopy

Electron microscopy was carried out on mature larvae. Larvae were fixedas before, post-fixed in 1% Osmium tetroxide and embedded in Spurrsresin. The blocks were orientated so that sections were cut from thedeveloping branchial basket area (an area potentially containing thecells of interest, as identified by in situ hybridisation) and sectionedusing an LKB ultramicrotome. Sections were mounted on grids and stainedusing uranyl acetate and lead citrate. Observations were made using aJeol I00S Transmission Electron Microscope.

Results:

The rods of the developing branchial basket in E turbinata larvae wereformed by elongate support cells. These were frequently vacuolated withlarge, electron dense inclusions (possibly lipids). Putative cells whichcorrelated with those observed by in situ hybridisation were identifiedand termed “bacteriocites” (FIG. 7). They had a very similar morphologyto the other major type of coelomocytes except for the presence ofputative bacteria. The bacteriocytes frequently had cytoplasmicprotrusions extending from the cell surface and all appeared to be freein the coelom. In small cells the nuclei were heterochromatic andunremarkable. Mitochondria were frequently observed. There were someobvious strands of rough endoplasmic reticulum, but the cells were mostnotable for the high densities of what are apparently polyribosomesdistributed throughout the cytoplasm. There was no obvious golgiapparatus.

The putative bacteria were rounded and approximately 1-2 microns indiameter. Their membrane structure suggests that they are gram-negativebacteria and they contain no obvious organelles (FIG. 7). Thechromosomal DNA is usually well distributed and only partiallycondensed. The bacteria appear to be healthy, particularly in thesmaller cells, with any obvious shrinkage probably the results of thefixation technique rather than any pathology. No bacteria were seenbeing lysed.

1. An isolated polynucleotide comprising or consisting of: (a) anucleotide sequence of SEQ ID NO: 1 or a modification, variant orfragment (part) thereof; (b) a nucleotide sequence having at least 50%identity to the nucleotide sequence of (a); (c) a nucleotide sequencecapable of hybridising to a polynucleotide according to (a) or (b);preferaby under stringent conditions, or (d) a fragment of apolynucleotide according to any one of (a) to (c).
 2. A polynucleotideaccording to claim 1, comprising or consisting of a nucleotide sequencehaving at least 70% identity with a polynucleotide according to (a), ora sequence hybridising therewith, or a fragment.
 3. A polynucleotideaccording to claim 1, comprising or consisting of a nucleotide sequencehaving at least 75% identity with a polynucleotide according to (a), ora sequence hybridising therewith, or a fragment.
 4. A polynucleotideaccording to claim 1, comprising or consisting of a nucleotide sequencehaving at least 85% identity with a polynucleotide according to (a), ora sequence hybridising therewith, or a fragment.
 5. A polynucleotideaccording to claim 1, comprising or consisting of a nucleotide sequencehaving at least 90% identity with a polynucleotide according to (a), ora sequence hybridising therewith, or a fragment.
 6. A polynucleotideaccording to claim 1, comprising or consisting of a nucleotide sequencehaving at least 95% identity with a polynucleotide according to (a), ora sequence hybridising therewith, or a fragment.
 7. A polynucleotideaccording to claim 1, comprising or consisting of a nucleotide sequencehaving at least 97% identity with a polynucleotide according to (a), ora sequence hybridising therewith, or a fragment.
 8. A polynucleotidefragment comprising or consisting of at least 5, 10, 15, 20, 25, 30 ormore contiguous nucleotides of a polynucleotide according to any one ofthe preceding claims.
 9. A probe or primer comprising or consisting of apolynucleotide fragment according to claim
 8. 10. A recombinant DNAcomprising or consisting of a polynucleotide sequence according to anyone of the preceeding claims.
 11. An isolated bacterium including apolynucleotide according to claim
 1. 12. An isolated bacterium accordingto claim 12 identified as Candidatus Endoecteinascidia frumentensis.